Cells that form the corneal epithelium, the outermost layer of the cornea, are exposed to shear stress through blinking during waking hours. In this in vitro study, the effect of fluid shear stress on human corneal epithelial cells (HCECs) was investigated. Following exposure to shear stresses of 4 and 8 dyn/cm2, HCECs showed cytoskeletal rearrangement with more prominent, organized and elongated filamentous actin. Cytoskeletal changes were time-dependent, and were most significant after 24 hours of shear stress. Higher rates of migration and proliferation, as evaluated by a scratch assay, were also observed following 24 hours of low shear stress exposure (4 dyn/cm2). This result contrasted the poor migration observed in samples scratched before shear exposure, indicating that shear-induced cytoskeletal changes played a key role in improved wound healing and must therefore precede any damage to the cell layer. HCEC cytoskeletal changes were accompanied by an upregulation in integrin β1 and downregulation of ICAM-1. These results demonstrate that HCECs respond favourably to flow-induced shear stress, impacting their proliferation and migration properties as well as phenotype.